In the last decade, mitochondria research has undergone tremendous progress as new technologies and increased understanding have paved the way for new areas of investigation. Mitochondria are organelles that provide an energy source for cell processes. New disciplines have emerged studying the role of the mitochondria in apoptosis and in human diseases. An increase in awareness of the role that mitochondrial dysfunction plays in human disorders, such as diabetes, neurological diseases, heart disease, and a host of other new discoveries of human disorders necessitated the development of a more effective method to isolate intact mitochondria.
The currently employed method for the isolation of intact mitochondria is conventionally referred to as dounce homogenization. In this method, cells are mechanically lysed to release cell components by homogenization in a Polytron tissue tearer or Dounce tissue grinder. The extent of lysis can be measured visually by comparison under a microscope of the original intact cells with those after douncing. Douncing is customarily accomplished under physiological conditions in a buffer containing sugars to preserve organelle integrity which does not swell the cell in order to minimize damage to the organelle during lysis.
Subsequent to cell lysis, the homogenate containing the mitochondria suspended in the buffer is centrifuged at low speed to remove unbroken cells, cellular debris and nuclei from the suspension. The supernatant is then centrifuged at high speed, and the mitochondria, in pellet form, are recovered, rinsed with the sugar containing buffer, and used for applications, including studies as indicated above related to apoptosis, signal transduction and metabolism.
In practicing methods for recovering mitochondria, the manipulative steps are accomplished at a temperature of less than about 10° C. in order to preserve the activity of the organelle. It is also customary to finely mince the tissue prior to beginning the recovery process.
Known douncing methods have been used to isolate mitochondria from cultured cells and hard tissue (e.g. muscle or heart) and soft tissue (e.g. liver or kidney). The methods are applicable with respect to mammalian cells as well as those from other species.